A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = Kagg(T−T0)2, where Kagg is the parameter characterizing the initial rate of aggregation and T0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets

Structural and Functional Peculiarities of Cytoplasmic Tropomyosin Isoforms, the Products of TPM1 and TPM4 Genes

Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are α-helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, TPM1 and TPM4, and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton

Structural basis for the ligand promiscuity of the neofunctionalized, carotenoid-binding fasciclin domain protein AstaP

Abstract Fasciclins (FAS1) are ancient adhesion protein domains with no common small ligand binding reported. A unique microalgal FAS1-containing astaxanthin (AXT)-binding protein (AstaP) binds a broad repertoire of carotenoids by a largely unknown mechanism. Here, we explain the ligand promiscuity of AstaP-orange1 (AstaPo1) by determining its NMR structure in complex with AXT and validating this structure by SAXS, calorimetry, optical spectroscopy and mutagenesis. α1-α2 helices of the AstaPo1 FAS1 domain embrace the carotenoid polyene like a jaw, forming a hydrophobic tunnel, too short to cap the AXT β-ionone rings and dictate specificity. AXT-contacting AstaPo1 residues exhibit different conservation in AstaPs with the tentative carotenoid-binding function and in FAS1 proteins generally, which supports the idea of AstaP neofunctionalization within green algae. Intriguingly, a cyanobacterial homolog with a similar domain structure cannot bind carotenoids under identical conditions. These structure-activity relationships provide the first step towards the sequence-based prediction of the carotenoid-binding FAS1 members

Effect of different agents on GDH aggregation.

<p>The initial parts of the dependences of the light scattering intensity on temperature for aggregation of GDH (0.12 mg/ml) in the presence of the following agents: (1) control; (2) 0.2 mM NADH; (3) 50 mM L-glutamate; (4) 0.2 mM NADH+0.5 mM ADP; (5) 50 mM L-leucine and (6) 0.5 mM ADP. Points are the experimental data. The solid curves were calculated from Eq. (4).</p

Effect of ADP on GDH aggregation.

<p>(<b>A</b>) The initial parts of the dependences of the light scattering intensity on temperature for aggregation of GDH (0.16 mg/ml) in the presence of the following concentrations of ADP: (1) 0, (2) 0.05, (3) 0.1, (4) 0.5 and (6) 2 mM. (<b>B</b> and <b>C</b>) The dependences of parameters <i>T</i>₀ and <i>K</i>_agg calculated from Eq. (4) on the ADP concentration, respectively. The solid curve in panel <b>C</b> was calculated from Eq. (10) at <i>K</i>_diss = 0.52 mM.</p

The relationships between the increment of the light scattering intensity accompanying Ph<i>b</i> aggregation and the portion of the denatured Ph<i>b</i> (γ_den) calculated from the DSC data.

<p>Ph<i>b</i> concentrations were as follows: (1) 0.95 and (2) 1.9 mg/ml. The inset shows the initial parts of the curves. Points are the experimental data. The solid curves are calculated from Eq. (6).</p

Estimation of the size of the protein aggregates formed in the course of CK aggregation registered in the regime of heating at the rate of 1°C/min (30 mM Hepes–NaOH buffer, pH 8.0, containing 0.1 M NaCl).

<p>(<b>A</b>) The initial parts of the dependences of the hydrodynamic radius (<i>R</i>_h) of the protein aggregates on temperature obtained at the following CK concentrations: (1) 0.1, (2) 0.75 and (3) 1.5 mg/ml. (<b>B</b> and <b>C</b>) The dependences of parameter <i>R</i>_h,0 and the reciprocal value of parameter Δ<i>T</i>_2R calculated from Eq. (9) on the GAPDH concentration, respectively.</p

Parameters of aggregation of GDH (0.12 mg/ml) in the presence of different ligands (0.1 M Na-phosphate buffer, pH 7.6).

<p>Parameters of aggregation of GDH (0.12 mg/ml) in the presence of different ligands (0.1 M Na-phosphate buffer, pH 7.6).</p

Effect of different agents on Ph<i>b</i> aggregation.

<p>The initial parts of the dependences of the light scattering intensity on temperature for aggregation of Ph<i>b</i> (0.5 mg/ml) in the presence of the following agents: (1) control, (2) 1 mM AMP, (3) 0.19 M HP-β-CD, (4) 1 M TMAO and (5) α-crystallin at the concentration of 1 mg/ml. Points are the experimental data. The solid curves were calculated from Eq. (4).</p